Generally, when live cells or microorganisms are examined to determine their characteristics they are placed under a microscope for analysis. Live cells are analyzed to find cures for many illnesses or diseases that exist today, such as cancer. For example, a person or scientist may put a breast lymph node cell on a specimen plate under a microscope to determine how the lymph node cell functions under various conditions in order to discover a method for treating the lymph node cell so it will not be cancerous.
A microscope that may be utilized to view cell function is a fluorescent microscope and the like. The typical fluorescent microscope utilizes a light source to transmit light through a dichroic mirror to excite fluorescent dyes in stained living cells or a sample specimen that absorbs radiation from the light and emits radiation at a lower frequency, whereby this emitted light will be reflected back through the dichroic mirror to an optical detector. The optical detector will then receive an image of the living cells. Normally, the optical detector will send the image to a computer that would reconstruct the image of the living cells based on an algorithm or equation.
Alternatively, one can use microscopy techniques other than fluorescence to view the cells, such as phase contrast microscopy, differential interference (DIC) microscopy, brightfield transmitted light microscopy and the like. Phase contrast microscopy is a contrast enhancing optical technique that can be utilized for generating high-contrast images of transparent specimens such as living cells, microorganisms and sub-cellular particles. This phase contrast technique employs an optical mechanism to translate minute variations in phase into corresponding changes in amplitude, which can be visualized as differences in image contrast. This type of microscopy enables one to observe low-contrast specimens that are either transparent or semi-transparent, which is often difficult, especially without proper illumination. The application of suitable contrast enhancement provides a substantial increase in contrast of barely visible low-contrast specimens in positive or negative relief. The illumination utilized by the phase contrast microscopy is standard brightfield transmitted light, oblique brightfield transmitted light and single-sided darkfield illumination. When a person utilizes standard brightfield transmitted light for illumination he avoids harmful exposure of the specimens to toxic dyes associated with staining living cells so the specimens will not die. However, the problem with utilizing this type of illumination is that brightfield images of the specimens look colorless and washed out. Moreover, in order to ensure that the specimen does not die it is necessary to keep the level of exposure the specimen receives from harmful light and bleaching to a minimum. Moreover, low intensity inevitably leads to noise being a severe problem.
Differential Interference Contrast (DIC) microscopy is a mechanism for increasing contrast in transparent specimens. DIC microscopy is a beam-shearing interference system in which the reference beam is sheared by a miniscule amount. This technique produces a monochromatic shadow-cast image that effectively displays the gradient of optical paths for both high and low spatial frequencies present in the specimen. The regions of the specimen where the optical paths increase along a reference direction appear brighter (or darker), while regions where the path differences decrease appear in reverse contrast. As the gradient of optical path difference grows steeper, image contrast is dramatically increased. Also, this type of microscopy enables one to observe low-contrast specimens that are either transparent or semi-transparent, which is often difficult especially without proper illumination. This DIC microscopy also utilizes standard brightfield transmitted light that causes the same problems discussed above for the phase contrast microscopy.
For brightfield transmitted light microscopes, light is aimed toward a lens beneath a stage called the condenser, through the sample specimen, through an objective lens, and to the eye through a second magnifying lens, the ocular or eyepiece. The object to be inspected is normally placed on a clear glass slide and light is transmitted through the object, which makes the object appear against a bright background hence the term “brightfield.” The objects in the light path are seen because natural pigmentation or stains absorb light differentially, or because they are thick enough to absorb a significant amount of light despite being colorless. The interior of the cells in the brightfield image is barely discernible so one can not tell the difference between the cells and the background. Also, the noise is a severe problem which inhibits segmentation of the cell. If one could segment cells in such brightfield images this would provide a wealth of information about cells that can be used as a diagnostic tool. For example, the utilization of the brightfield imaging technique is very useful in cancer research because this technique allows cancer cells to be kept alive, which is necessary in order to perform cancer research. On the other hand, when other imaging techniques are utilized living cells are killed when they are stained, which prohibits scanning of cells for cancer research.
In order to detect, diagnose and treat living cells in brightfield images these cells, such as cancer cells must be analyzed by segmenting and reconstructing the image of living cells. Therefore, there is a need for a system that is able to analyze living cells in brightfield images where the living cells can be discerned from the background of the sample specimen.